A method for isolating adult and neonatal airway smooth muscle cells and measuring shortening velocity

Methods are described for isolating smooth muscle cells from the tracheae o f adult and neonatal sheep and measuring the single-cell shortening velocit y. Isolated cells were elongated, Ca2+ tolerant, and contracted rapidly and substantially when exposed to cholinergic agonists, KCl serotonin, or caff eine. Adult cells were longer and wider than preterm cells. Mean cell lengt h in 1.6 mM CaCl2 was 194 +/- 57 (SD) mu m (n = 66) for adult cells and 93 +/- 32 mu m (n = 20) for preterm cells (P < 0.05). Mean cell width at the w idest point of the adult cells was 8.2 +/- 1.8 mu m (n = 66) and 5.2 +/- 1. 5 mu m (n = 20) for preterm cells (P < 0.05). Cells were loaded into a perf usion dish maintained at 35 degrees C and exposed to agonists, and contract ions were videotaped. Cell lengths were measured from 30 video frames and p lotted as a function of time. Nonlinear fitting of cell length to an expone ntial model gave shortening velocities faster than most of those reported f or airway smooth muscle tissues. For a sample of 10 adult and 10 preterm ce lls stimulated with 100 mu M carbachol, mean (+/- SD) shortening velocity o f the preterm cells was not different from that of the adult cells (0.64 +/ - 0.30 vs. 0.54 +/- 0.27 s(-1), respectively), but preterm cells shortened more than adult cells (68 +/- 12 vs. 55 +/- 11% of starting length, respect ively; P < 0.05). The preparative and analytic methods described here are w idely applicable to other smooth muscles and will allow contraction to be s tudied quantitatively at the single-cell level.